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Solutions
Stock Solutions HCl, 1M, 50ml *Add 4.31ml concentrated HCl to 45.69ml H20 *NaCl, 5M, 100ml *29.2g NaCl *100ul DMPC *H2O to 100ml *Mix and autoclave NaOH, 5M, 50ml *10 g NaOH in 25 ml H2O *Add H2O to 50 ml LiCl, 10M, 50ml *21 g LiCl in 25ml H2O *50 ul DMPC *Add H2O to 50 ml *Mix and autoclave SDS, 10%, 50 ml ' Pour slowly to prevent bubbling *5 g SDS in 25 ml H2O *Add H2O to 50 ml '''CTAB, 20%, 50 ml ' Pour slowly to prevent bubbling *10 g CTAB in 25 ml H2O *Add 50 ul DMPC *Add H2O to 50 ml *Mix and autoclave 'PVP-40, 20%, 50 ml ' Pour slowly to prevent bubbling *10 g PVP-40 in 25 ml H20 *Add 50 ul DMPC *Add H2O to 50 ml *Mix and autoclave '''EDTA, 0.5M, pH 8, 50 ml *Dissolve 9.3 g Na2EDTA-2H2O in 35 ml H2O *Adjust to pH8 with 10M NaOH (~2.5 ml) *Add 50 ul DMPC *Add H2O to 50 ml *Mix and autoclave TRIS-HCl, 1M, pH 8, 50 ml *6.05 g Tris base in 40 ml H2O *Adjust to pH 8 with concentrated HCl *Add H2O to 50 ml and mix Spermidine, 50 g/L, 5 ml *Add 0.25 g spermidine to 4 ml H2O *Add H2O to 5 ml *Sterilize by filtering CTAB Extraction buffer, 100 ml *10 ml CTAB (20%) *10 ml PVP-40 (20%) *10 ml Tris-HCl (1M) *5 ml EDTA (0.5M) *40 ml NaCl (5M) *Add H2O to 97 ml *Mix and autoclave *1 ml filter purified spermidine *2 ml B-mercaptoethanol (just before use) SSTE buffer, 50 ml *10 ml NaCl (5M) *2.5 ml SDS (10%) *0.5 ml Tris-HCl (1M, pH 8) *0.1 ml EDTA (0.5M, pH 8) *Mix and autoclave Chloroform-isoamyl alcohol 24:1, 50 ml *Add 2 ml isoamyl alcohol to 48 ml chloroform 15% PEG 8000 – 2M NaCl, 50 ml *Add 7.5 g PEG 8000 to 5.84g NaCl in 25 ml H2O *Add 50 ul DMPC *Add H2O to 50 ml *Mix and autoclave 10X loading buffer *20% Ficoll 400 *0.1M Na2EDTA, pH 8 *1.0% sodium dodecyl sulphate *0.25% bromophenol blue *0.25% xylene cyanol Solutions for RNA extraction *'HCl, 1M, 50 ml' **Add 4.32 ml concentrated HCl to 45.69 ml H2O *'NaCl, 5M, 100 ml' **Dissolve 29.2 g NaCl in 75 ml H2O **Add 100 ul DMPC **Add H2O to 100 ml **Mix and autoclave *'NaOH, 5M, 50 ml' **Dissolve 10 g NaOH in 30 ml H2O **Add H2O to 50 ml *'LiCl, 10M, 50 ml' **Dissolve 21 g LiCl in 30 ml of water (this will take at quite a while) **Add H2O to 50 ml **Sterilize by filtration *'SDS, 10%, 50 ml' **Dissolve 5 g SDS in 30 ml water mixing slowly to avoid bubbling **Add H2O to 50 ml pouring slowly to avoid bubbling *'CTAB, 20%, 50 ml' **Dissolve 10 g CTAB in 30 ml H2O mixing slowly to avoid bubbling **Add 50 ul DMPC **Add H2O to 50 ml **Mix and autoclave *'PVP-40, 20%, 50 ml' **Dissolve 10 g PVP-40 in 30 ml H2O mixing slowly to avoid bubbling **Add H2O to 50 ml *'EDTA, 0.5M, pH 8, 50 ml' **Dissolve 9.3 g Na2EDTA-2H2O in 35 ml H2O **Adjust to pH 8 with 10M NaOH (~2.5ml) **Add 50 ul DMPC **Add H2O to 50 ml **Mix and autoclave *'Tris-HCl, 1M, pH 8, 50 ml' **Dissolve 6.05 g Tris base in 30 ml H2O **Adjust to pH 8 with concentrated HCl **Add H2O to 50 ml **Mix and autoclave *'Spermidine, 50 g/L, 10 ml' **Add 0.5 g spermidine to 8 ml H2O **Add H2O to 5 ml **Sterilize by filtration *'CTAB extraction buffer, 100 ml' **10 ml CTAB (20%) **10 ml PVP-40 (20%) **10 ml Tris-HCl (1M) **5 ml EDTA (0.5M) **40 ml NaCl (5M) **Add H2O to 97 ml **Mix and autoclave **Add 1 ml filter purified spermidine (50 g/L) **Add 3% B-mercaptoethanol immediately prior to use Solutions for preparing plates *''IPTG, 0.1M'' **1.2g IPTG **Add water to 50 ml final volume **Filter sterilize and store at 4 degrees C *'X-Gal, 50 mg/ml' **100 mg 5-bromo-4-chloro-3-indolyl-B-D-galactoside **Dissolve in 2 ml N,N'-dimethyl-formamide **Cover with aluminum foil and store at -20 degrees C *'Ampicillin, 100 mg/ml' **1.0 g Ampicillin **Dissolve in 10 ml sterile water **Store at -20 degrees C **Ampicillin is sensitive to light so try to avoid exposure *'LB medium' **10 g LB broth **Add deionized water to 500 ml **Adjust to pH 7.0 with NaOH **If you aren't using the LB medium to make LB plates then autoclave it *'LB plates with ampicillin/IPTG/X-Gal' **Add 7.5 g agar to 500 ml of LB medium. Mix and autoclave. Allow the medium to cool to 50 degrees C before adding the following: ***500 ul ampicillin (100 mg/ml) ***2.5 ml IPTG (0.1 M) ***800 ul X-Gal (50 mg/ml) **For each plate pour a small dollop of medium into the center of an 85 mm petri dish. **Gently swirl the dish until the medium forms a complete layer covering the bottom of the dish. **Let the agar harden. **Store at 4 degrees C for up to 1 month Category:Solutions